Ann Surg Oncol (2010) 17:3362–3369 DOI 10.1245/s10434-010-1149-2

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Serum Matrix-Metalloproteinase-1 is a Bona Fide Prognostic Marker for Colorectal Cancer Kouichirou Tahara, MD, PhD1, Koshi Mimori, MD, PhD1, Hisae Iinuma, PhD2, Masaaki Iwatsuki, MD, PhD1, Takehiko Yokobori, MD1, Hideshi Ishii, MD, PhD1, Hideaki Anai, MD, PhD3, Seigo Kitano, MD, PhD4, and Masaki Mori, MD, PhD, FACS5 Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan; 2Department of Surgery, Teikyo University School of Medicine, Tokyo, Japan; 3Department of Surgery, Oita Medical Center, Oita, Japan; 4 Department of Surgery I, Faculty of Medicine, Oita University, Oita, Japan; 5Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Japan 1

ABSTRACT Background. Matrix metalloproteinases (MMPs) are involved in the degradation of extracellular matrix components and are associated with invasion and metastasis. MMP proteins could be serum tumor markers or molecular targets in the treatment of malignancy. The purpose of the current study was to identify a prognostic serum marker in cases of colorectal cancer (CRC) prior to surgical intervention. Materials and Methods. Laser microdissection and microarray analysis were used to characterize gene expression in 73 cases of CRC. We then focused on expression of MMP-1. We examined serum MMP-1 activity before resection in another subset of 75 cases of CRC to validate the clinical significance of MMP-1 as a prognostic marker in CRC after surgically curative operation. Results. Disease-free survival was 51% in the MMP-1 high expression group and 81% in the low-expression group (P \ .05). Survival was 52% in the MMP-1 high expression group and 90% in the low group (P \ .05). In multivariate analysis for disease-free survival, MMP-1 and lymph node metastasis were significant independent prognostic indicators. In multivariate analysis of overall survival, serum MMP-1 level was the only significant independent indicator among factors.

Kouichirou Tahara and Koshi Mimori have contributed equally to this work. Ó Society of Surgical Oncology 2010 First Received: 13 July 2009; Published Online: 9 July 2010 M. Mori, MD, PhD, FACS e-mail: [email protected]

Conclusions. Within the MMP family of proteins, MMP-1 is not a cancer-specific protease. However, MMP-1 activity does predict the future course of progression of malignant cells. Thus, MMP-1, which is activated at the primary lesion and is found in serum, assists in the clinical diagnosis of CRC. It is also an important molecule for understanding the underlying mechanism of invasion and metastasis of CRC.

In colorectal cancer (CRC), several prognostic factors have been identified, including clinicopathologic status, and gene and protein expression at primary tumor sites. Unfortunately, there are few indicators that reliably and specifically predict recurrence, metastasis, and long-term prognosis in CRC. It would be advantageous if it were possible to repeatedly assess patients’ tumor markers on an outpatient basis without use of invasive techniques. In that regard, serum tumor markers would be particularly useful. Over the past half century, serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 have become well-established prognostic indicators in CRC.1,2 However, no tumor marker is generally accepted as optimal in its prognostic power. Therefore, we attempted to identify novel and reliable prognostic markers within serum that could predict both tumor recurrence and metastasis. Matrix metalloproteases (MMPs) are the most important members of a multigene family comprising more than 25 related zinc-dependent enzymes involved in the degradation of extracellular matrix components in CRC. MMP-1, MMP2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, and MT1MMP have been studied extensively in CRC.3 In particular, MMP-2 and MMP-9 show high expression at cancer sites,

Serum MMP-1 in Colorectal Cancer

and their expression is correlated with Dukes staging.4,5 Moreover, MMP-7 is expressed at almost all CRC sites.6,7 There has been unanimous agreement that enhanced expression of MMP-7 in CRC correlates with the presence of nodal or distant metastasis.3 MMP-1, MMP-2, MMP-7, MMP-9, and MMP-11 may play roles in both conversion from adenoma to carcinoma and in the initiation of invasion and metastasis.8 Thus, these MMPs have been regarded as particularly important in cancer progression. Several MMPs might constitute good prognostic markers in CRC because they are secreted as inactive zymogens, are activated extracellularly, and can be measured in the serum.9,10 Serum MMP-2, MMP-9, and MMP-7 in CRC patients have already been analyzed, and there was a clear correlation between serum MMP-7 level and poor prognosis.11 On the other hand, matrilysin, specifically degrades collagen types I, II, and III, which are the main components of the interstitial stroma.11,12 Increased expression of MMP-1 has been observed in CRC, and increased collagenolytic activity correlates with poor differentiation of the tumors. Therefore, we anticipated that serum levels of MMP-1 would constitute an important prognostic marker. MATERIALS AND METHODS Microarray Analysis of Patients’ Tissue We examined gene expression profiles of CRC-specific genes in 73 CRC patients from the Department of Surgery, Medical Institute of Bioregulation, Kyushu University. We used laser microdissection (LMD) for collection of cancer cells, stromal cells, and normal cells to perform microarray analysis. Based on microarray analyses, we selected the best MMP as a candidate tumor marker. We excluded MMP-2, MMP-9, and MMP-7 from further consideration because those MMPs had already been studied in earlier reports. Serum Analysis of Patients MMP-1was examined in the serum of another subset of CRC patients. This study used serial collection of serum samples from 100 CRC patients who underwent primary surgery from July 2000 to December 2004 in the Department of Surgery, Teikyo University. The patients were in good condition (performance status (PS) B 2) and were suitable for resection of their primary tumor. All patients had a medical history, clinical examination, full blood count, and a biochemical screen of renal and liver function. Staging was done by abdominal computed tomography (CT) and chest radiography. Additional techniques such as abdominal ultrasound, chest CT, or magnetic resonance imaging (MRI) were used if needed for further refinement

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of staging. Serum samples were obtained before surgery, after documented informed consent. Collection of Target Cells by LMD from Frozen Sections For LMD, frozen section slides were fixed in 70% ethanol for 30 s, stained with hematoxylin and eosin, and the following dehydration steps were performed: 5 s each in 70, 95, and 100% ethanol and air-dried. Once dry, the sections were laser microdissected with the LMD system. Target cells were excised, at least 500–1,500 cells per section, and bound to the transfer film. A total of 15 sections were collected from every sample, equivalent to approximately 10,000–15,000 cells per sample. RNA Extraction and the Oligonucleotide Microarray The cells collected with LMD were placed in a microcentrifuge tube with 350 ll RLT buffer containing 1% 2mercaptoethanol. Total RNA was extracted using the RNeasy Micro Kit (QIAGEN, Valencia) according to the manufacturer’s protocol. The concentration and purity of the RNA were determined with the Nano Drop (Nano Drop Technologies, Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), respectively, as described previously. We used the commercially available Human Whole Genome Oligo Microarray Kit (Agilent Technologies) that contained more than 41,000 features, including 36,866 characterized human genes. Cyaninelabeled cRNA was prepared using T7 linear amplification as described in the Agilent Low RNA Input Fluorescent Linear Amplification Kit Manual (Agilent Technologies). Briefly, 100 ng of purified total RNA was reverse transcribed to generate double-stranded cDNA using an oligo dT T7 promoter primer and MMLV reverse transcriptase. Next, cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated Cy 3 or Cy 5 labeled CTP. During this process, the samples of cancer cells were labeled with Cy 5, whereas the Human Universal Reference Total RNA (BD Clontech, Palo Alto) was labeled with Cy 3 as a control. Quality of the cRNA was verified with the Agilent 2100 Bioanalyzer. One lg aliquots each of Cy 3 labeled cRNA and Cy 5 labeled cRNA were combined and fragmented in a hybridization cocktail (Agilent Technologies). The labeled cRNAs were then hybridized to a 60-mer probe oligonucleotide microarray and incubated for 17 h at 60°C. The fluorescent intensities were determined by an Agilent DNA Microarray Scanner and were analyzed by G2567AA Feature Extraction Software Version A.7.5.1 (Agilent Technologies). This software uses the LOWESS (locally weighted linear regression curve fit) normalization method. This microarray study followed

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MIAME guidelines issued by the Microarray Gene Expression Data group. Further analyses were done using GeneSpring version 7.3 (Silicon Genetics, San Carlos, CA). Microarray data were available from our previous study (http://cibex.nig.ac.jp/cibex2/index.jsp).13

A disease-free survival curve and overall survival curve were plotted according to the Kaplan–Meier method. The difference between the survival curves was analyzed by the Wilcoxon and log-rank test. The effects of various clinicopathological factors on disease-free and overall survival (including the serum level of MMP-1) were assessed by the Cox proportional hazards model. All analyses were performed using Stat View 5.0 for Windows (SAS Institute, Cary, NC). The study protocol was approved by the Institutional Review Board, and informed consent was obtained from all patients.

Serum Collection At 5 min prior to surgery, venous blood samples were drawn into sterile vacuum tubes and left at room temperature for 30 min. They were then centrifuged at 1,500 rpm for 15 min. Serum was immediately aliquoted and kept at -80°C until assayed.

RESULTS Gene Expression Within the MMP Family in CRC and Selection of Candidate Markers for Serum Analysis

Serum Samples and MMP-1 Analysis MMP-1 was determined using a quantitative solid-phase sandwich enzyme linked immunosorbent assay (ELISA) and tested in duplicate.14,15 We also examined serum MT1MMP by ELISA.16

Figure 1 shows the expression of all MMP family genes in stromal cells and cancer cells excised by laser microdissection. These data show the mean values from cancer cells and normal cells in colon epithelial tissues from 73 CRC patients. In addition, normal cells from non-CRC patients were analyzed at the same time, and the normal level of gene expression was set at 1.00 for each MMP. In cancer cells, expression values for MMP-1, MMP-3, MMP7, MMP-11, and MMP-12 were 3 times higher than those observed in normal cells. In fact, expression of those MMPs was elevated in interstitial cells as well as in cancer cells in CRC. In addition, expression levels of MMP-2, MMP-9, and MMP-10 in stromal cells were more than 10 times higher than in normal controls. Other MMPs showed

Statistical Methods Recorded variables included age, gender, date of surgery, date of death or last follow-up, histological records such as site of primary cancer, tumor size, depth of invasion, lymph node metastasis, lymphatic invasion, venous invasion and Dukes’ stage, and serum level of MMP-1, as described previously. Missing values (\2%) were omitted from multivariate analyses.

a 35 30 25 20 15 10 5

b 90 80 70 60 50 40 30 20

7

P2 8

P2

M

M

M

M

5

P2 6

P2

M

M M

M

1

B

P2

P2 3

M

M

M

M M

M

9

P2 0

7

P1

P1

M

M

M

M

5

P1 6

P1

M

M

M

M

3

P1 4 M

M

M

M

P1

2

1

P1 M

M

M

M

P1

P1 0

P9

M M

P8

M M

P7

M

M

P3

M M

P2

M M

M

M

M

P1

10

M

FIG. 1 Relative expression of MMPs in interstitial cells adjacent to cancer tissues (a) and in cancer cells (b). a The vertical axis showed the relative expression ratio of MMPs in interstitial cells in comparison to that in normal control cells. MMP-1, MMP-3, MMP-7, MMP-11, and MMP-12 were three times higher than normal controls. b In addition to the above 5 MMPs, expression levels of MMP-2, MMP-9, and MMP-10 were more than 10 times higher than normal controls. Therefore, expression of MMP-1, MMP-3, MMP-7, MMP-11, and MMP-12 (colored red) was elevated in interstitial cells as well as in cancer cells in CRC. Other MMPs (colored blue) showed little expression in this microarray analysis

Serum MMP-1 in Colorectal Cancer

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little expression in this microarray analysis. These data identified MMP-1, MMP-3, MMP-11, and MMP-12 as candidates for further serum analysis. Although MMP-11 seemed to be an attractive candidate because of its high expression values, we focused on MMP-1 because this enzyme seems to play a more important role than the other 3 MMPs both in the transition from adenoma to carcinoma, and CRC progression as well as positive interactions of MMP-2, MMP-9, and MMP-7.8 MMP-11 seems to contribute only to the transition from adenoma to carcinoma.8

The correlations between serum MMP-1 levels and clinicopathological factors, including recurrence, are shown in Table 1. Although no significant association was found for pathological factors, serum MMP-1 levels were higher in patients with recurrence of disease. In addition, the serum levels of MT1-MMP (MMP-14) and CEA were compared with clinicopathologic variables. No significant association was found. However, the incidence of recurrence was higher in serum MT1-MMP-positive cases (Table 1, P \ .05).

Patient Characteristics and Serum Levels of MMP-1 in CRC Patients Receiving Curative Resection

Association of Disease-Free Survival and Overall Survival with Serum MMP-1 Levels in CRC Patients Undergoing Curative Resection

During the study period, 100 patients with CRC were diagnosed for the first time and underwent colectomy at Teikyo University. Within this group, 25 patients were found to be surgically noncurative, and 75 patients were potentially surgically curative and were approved for the current study. Median age of the patients in our sample was 68.6 years, and 57.3% were males. The mean and median serum levels of MMP-1 were 49.2 and 48 ng/ml, respectively (range 8–161 ng/ml).

The cut-off value for serum MMP-1 levels was set at the average value (49.2 ng/ml), and patients were divided into 2 groups, ‘‘MMP-1 high’’ and ‘‘MMP-1 low.’’ The MMP-1 high group consisted of 25 patients, and the low group contained 50 patients. We divided 75 patients by the average expression level of MMP-1. Disease-free survival was 51% in the MMP-1 high group and 81% in the low group (P \ .05) (Fig. 2). Overall survival was 52% in the

TABLE 1 Serum MMP1 and MT1-MMP levels and correlation with clinicopathologic factors n

Serum MMP1 (ng/ml)

Shallow

27

46.3 ? 7.4

Deep

48

49.3 ? 5.5

Depth of tumor

P value

Serum MT1-MMP (ng/ml)

ns

Histology

P value

Serum CEA (ng/ml)

ns 0.56 ? 0.14

Serum CA19-9 (ng/ml)

ns 6.3 ? 11.2

0.67 ? 0.1 ns

P value

ns 18.3 ? 24.5

24.4 ? 8.4 ns

79.2 ? 18.4 ns

ns

Well

56

49.2 ? 5.1

0.45 ? 0.17

13.3 ? 7.8

71.4 ? 30.0

Mod

19

45.4 ? 8.8

0.70 ? 0.10

31.4 ? 12.4

52.5 ? 17.4

\5.0 cm

45

53.5 ? 5.6

0.51 ? 0.11

13.7 ? 8.7

18.1 ? 18.1

[5.0 cm

30

40.3 ? 6.9

0.82 ? 0.13

24.1 ? 10.7

116.1 ? 22.2

Absent

54

50.5 ? 5.2

Present

21

42.4 ? 8.3

Tumor size

ns

Lymphatic permeation

ns

Vascular permeation Absent Present

ns

47.7 ? 6.1 48.8 ? 6.5

Absent

50

47.8 ? 5.4

Present

25

49.2 ? 7.7

Lymph node metastasis

ns

Recurrence

ns

ns 40.6 ? 20.6 76.4 ? 21.9

ns 14.5 ? 8.3

0.77 ? 0.15 .0013

57.1 ? 28.6 ns

15.1 ? 8.0 24.9 ? 12.8

0.56 ? 0.10

ns 57.4 ? 17.6

24.9 ? 12.8 ns

0.54 ? 0.12 0.73 ? 0.12

.001

ns 15.1 ? 8.0

0.72 ? 0.16 ns

40 35

ns

ns 0.60 ? 0.10

.0002 19.6 ? 16.9

24.5 ? 11.7 .018

P value

132.8 ? 23.9 ns

.0007

Absent

54

39.6 ? 4.9

0.51 ? 0.10

13.9 ? 8.0

26.3 ? 16.5

Present

21

70.4 ? 7.8

0.95 ? 0.16

28.1 ? 12.8

137.0 ? 26.4

n number of cases, ns no statistical significance, shallow tumor invading within proper muscle, deep tumor invading beyond the proper muscle, well well differentiated adenocarcinoma, mod moderately differentiated adenocarcinoma

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MMP-1 high group and 90% in the low group (P \ .05) (Fig. 3). In addition, serum MT1-MMP, CEA, and CA19-9 were analyzed in the same way. There were no statistically significant correlations for MT1-MMP and CEA values. However, high CA19-9 activity indicated a higher incidence of recurrence than did CA19-9 low activity (Figs. 2, 3). Univariate analysis of disease-free survival showed significant differences in MMP-1 level, depth of invasion and lymph node metastasis (Table 2). Univariate analysis for overall survival showed significant differences in MMP-1 level, depth of invasion, lymph node metastasis and venous invasion (Table 3). In multivariate analysis of disease-free survival, MMP-1 level and lymph node metastasis were significant independent prognostic indicators (Table 2). In multivariate analysis of overall survival, MMP-1 level was the only significant independent prognostic indicator (Table 3). Serum MT1-MMP was not a significant independent prognostic factor for disease-free survival or overall survival in univariate and multivariate analyses (Tables 2, 3).

FIG. 2 Disease-free survival rates and correlations with serum markers in 75 cases of CRC following curative surgery. a There were significant differences between the 25 cases with high MMP-1 serum activity versus the 50 cases with low MMP-1 serum activity (log rank P = 0.025, Wilcoxon P = .036). b No significant difference was observed between 39 cases with MT1-MMP high activity and 36 cases with low activity. c There was no statistical significance in CEA values; however, 18 cases with high CA19-9 activity indicated a higher incidence of recurrence than the 57 cases with low CA19-9 activity (log rank P = .012, Wilcoxon P = .028)

DISCUSSION In the current study, we examined serum MMP levels to determine which was most suitable to predict the course of disease in CRC cases.11,17 Microarray analyses showed that MMP-1, MMP-3, MMP-7, MMP-11, and MMP-12 were overexpressed in both cancer cells and stromal cells. Among these, we focused on MMP-1 and demonstrated that high levels of serum MMP-1 correlated with poor survival in patients with CRC after curative resection. Furthermore, serum MMP-1 was an independent and specific prognostic factor for survival in CRC patients. Moreover, we showed that serum MMP-1 level was more useful than more established serum tumor markers, such as CEA and CA19-9, as shown in Figs. 2, 3. There is substantial evidence that MMP-2, MMP-9, and MMP-7 are overexpressed in primary CRC tumors. MMP-7 contributed broadly to each stage of CRC progression: transition from normal mucosa to adenoma, and adenoma to carcinoma, and then to metastasis. Recently, it was reported that serum MMP-7 levels are significantly elevated in patients with

a

b

Disease Free Survival 1.0

Disease Free Survival 1.0

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4 0.3 0.2 0.1

0.4

MMP1 High (n=25) MMP1 Low (n=50)

0.3 0.2

Log rank p=0.025 Wilcoxon p=0.036 1

0.1

2

3

4

5

MT1-MMP1 High (n=36) MT1-MMP1 Low (n=39) Log rank n.s. Wilcoxon n.s. 1

2

Years

c

d

Disease Free Survival 1.0

Disease Free Survival 1.0

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4 0.3 0.2 0.1

0.4

CEA High (n=25) CEA Low (n=50)

0.3 0.2

Log rank n.s. Wilcoxon n.s. 1

0.1 2

3

Years

3

4

5

3

4

5

Years

4

5

CA 19-9 High (n=18) CA 19-9 Low (n=57) Log rank p=0.012 Wilcoxon p=0.028 1

2

Years

Serum MMP-1 in Colorectal Cancer FIG. 3 Overall survival (OS) rates and correlations with serum markers in 75 cases of CRC following curative surgery. a 25 cases with high MMP-1 activity showed poorer OS than 50 cases with low MMP-1 activity (log rank P = .013, Wilcoxon P = .029). However, there were no statistically significant differences in b serum MT1MMP, c CEA, or d CA19-9

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a

b

Overall Survival 1.0

Overall Survival 1.0

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4 0.3 0.2 0.1

0.4

MMP1 High (n=25) MMP1 Low (n=50)

0.2

Log rank p=0.013 Wilcoxon p=0.029 1

Log rank n.s. Wilcoxon n.s.

0.1

2

3

4

5

Years

c

MT1-MMP1 High (n=36) MT1-MMP1 Low (n=39)

0.3

1

d Overall Survival 1.0

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.3 0.2 0.1

0.4

CEA High (n=25) CEA Low (n=50)

1

0.2

3

4

5

3

4

5

Log rank n.s. Wilcoxon n.s.

0.1 2

4

CA 19-9 High (n=18) CA 19-9 Low (n=57)

0.3

Log rank n.s. Wilcoxon n.s.

3

Years

Overall Survival 1.0

0.4

2

1

5

2

Years

Years

TABLE 2 Univariate and multivariate analysis for disease free survival in CRC cases with curative operation Univariate analysis RC

P value

Hazard ratio

RC

Serum MMP1

0.488

1.629

.027*

Serum MT1-MMP

0.628

1.874

.084

Depth of tumor

Multivariate analysis

0.473

-0.744

0.475

.006*

-0.493

Lymph node metastasis

0.617

1.853

.006*

0.467

Histology

0.12

1.128

.625

-0.315

0.729

.151

0.237 0.368

1.268 1.444

.301 .967

Tumor size Lymphatic permeation Vascular permeation

P value

Hazard ratio 1.605

.033*

0.61

.12

1.594

.047*

* There is a statistical significance

CRC and serum MMP-7 is an independent prognostic factor for survival in advanced CRC.11 On the other hand, MMP-2, and MMP-9 may be most important in the early steps of CRC carcinogenesis. Recent studies suggest that serum type IV collagenase (MMP-2 and MMP-9) may be correlated with progression of CRC; however, it has not been proven that serum MMP-2 and MMP-9 are preferred prognostic indicators.

As for the origin of serum MMP-1, there are 2 possibilities. Cancer cells and host stromal cells secrete MMP-1 at the primary cancer site. During tumor progression and invasion, MMP-1 might then drain into the circulation. Alternatively, MMP-1 might be secreted as a response against the invasion of cancer cells, not only at the primary cancer site but also in bone marrow or peripheral blood. The serum MMP-1 level, then, likely represents the total

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TABLE 3 Univariate and multivariate analysis for overall survival in CRC cases with curative operation Univariate analysis RC

P value

Hazard ratio

RC

Serum MMP1

0.693

1.998

.014*

Serum MT1-MMP

0.182

1.2

.74

-0.721

0.486

.028*

Lymph node metastasis

0.61

1.841

.025*

Histology

0.087

1.091

.772

-0.112

0.894

.68

0.386 0.576

1.471 1.78

.17 .039*

Depth of tumor

Tumor size Lymphatic permeation Vascular permeation

Multivariate analysis

0.667

P value

Hazard ratio 1.95

.019*

-0.163

0.849

.732

0.529

1.698

.07

0.355

1.426

.28

* There is a statistical significance

amount of MMP-1 secreted from host tissues. On the other hand, Trivedi et al. suggested that MMP-1 might be derived from peripheral blood platelets.18 Platelets harbor several MMPs that modulate hemostatic function and platelet survival, and they demonstrated that platelet MMP1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. MMP-1 mediated aggregation through PAR1 activates Rho-GTP pathways, cell shape change, motility, and MAPK signaling.18 Indeed, we speculate that platelets activated by MMP-1 might play an important role in cancer metastasis. Further investigation will be required to identify the origin of MMP-1 in peripheral blood. Most MMPs are secreted as inactive zymogens and are activated extracellularly. A portion of the MMP family (MT1-MMP, MMP-16, MMP-17, MMP-24, and MMP-25) is constituted by transmembrane proteases that are activated on the cell surface.19,20 These MMPs may be good tumor markers because of their tumor specificity.21 The current microarray expression profile showed that most of these transmembrane MMPs were expressed at low levels. Of the transmembrane MMPs, MT1-MMP showed the highest gene expression level. However, the serum level of MT1-MMP was not correlated with CRC patient prognosis. It appears that the expression level of MT1-MMP was too low to show significant differences in the serum. In conclusion, within the MMP family, MMP-1 is not a cancer-specific protease. However, its expression level predicts the course of invasion and progression of malignant cells when assessed in the serum of CRC patients. In this study, the number of enrolled patients was small and the timing of sample measurement was necessarily limited to a single point. Further investigation is needed to validate the reproducibility of the current data. ACKNOWLEDGMENT We thank T. Shimooka, K. Ogata, M. Kasagi, Y. Nakagawa, and T. Kawano for their technical assistance. This work was supported in part by the following grants and foundations: CREST, Japan Science and Technology Agency (JST); Japan

Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research, grant No. 20012039, 20390360, 20590313, 20591547, 20659209, 20790960, 21591644, 21592014, 21791295, 21791297, 215921014, 21229015, and 21679006; NEDO (New Energy and Industrial Technology Development Organization) Technological Development for Chromosome Analysis; Grant of Clinical Research Foundation (2008–2010).

REFERENCES 1. Duffy MJ, van Dalen A, Haglund C, Hansson L, Holinski-Feder E, Klapdor R, et al. Tumour markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines for clinical use. Eur J Cancer. 2007;43:1348–60. 2. Woolfson K. Tumor markers in cancer of the colon and rectum. Dis Colon Rectum. 1991;34:506–11. 3. Zucker S, Vacirca J. Role of matrix metalloproteinases (MMPs) in colorectal cancer. Cancer Metastasis Rev. 2004;23:101–17. 4. Ring P, Johansson K, Hoyhtya M, Rubin K, Lindmark G. Expression of tissue inhibitor of metalloproteinases TIMP-2 in human colorectal cancer—a predictor of tumour stage. Br J Cancer. 1997;76:805–11. 5. Zeng ZS, Huang Y, Cohen AM, Guillem JG. Prediction of colorectal cancer relapse and survival via tissue RNA levels of matrix metalloproteinase-9. J Clin Oncol. 1996;14:3133–40. 6. Mori M, Barnard GF, Mimori K, Ueo H, Akiyoshi T, Sugimachi K. Overexpression of matrix metalloproteinase-7 mRNA in human colon carcinomas. Cancer. 1995;75:1516–9. 7. Yoshimoto M, Itoh F, Yamamoto H, Hinoda Y, Imai K, Yachi A. Expression of MMP-7(PUMP-1) mRNA in human colorectal cancers. Int J Cancer. 1993;54:614–8. 8. Soreide K, Janssen EA, Korner H, Baak JP. Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis. J Pathol. 2006;209:147–56. 9. Zucker S, Lysik RM, Zarrabi MH, Moll U. M(r) 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res. 1993;53:140–6. 10. Langenskiold M, Holmdahl L, Falk P, Ivarsson ML. Increased plasma MMP-2 protein expression in lymph node-positive patients with colorectal cancer. Int J Colorectal Dis. 2005; 20:245–52. 11. Maurel J, Nadal C, Garcia-Albeniz X, Gallego R, Carcereny E, Almendro V, et al. Serum matrix metalloproteinase 7 levels identifies poor prognosis advanced colorectal cancer patients. Int J Cancer. 2007;121:1066–71.

Serum MMP-1 in Colorectal Cancer 12. Murray GI, Duncan ME, O’Neil P, Melvin WT, Fothergill JE. Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer. Nat Med. 1996;2:461–2. 13. Yokoe T, Tanaka F, Mimori K, Inoue H, Ohmachi T, Kusunoki M, et al. Efficient identification of a novel cancer/testis antigen for immunotherapy using three-step microarray analysis. Cancer Res. 2008;68:1074–82. 14. Green MJ, Gough AK, Devlin J, Smith J, Astin P, Taylor D, et al. Serum MMP-3 and MMP-1 and progression of joint damage in early rheumatoid arthritis. Rheumatology (Oxford). 2003;42: 83–8. 15. Myers A, Lakey R, Cawston TE, Kay LJ, Walker DJ. Serum MMP-1 and TIMP-1 levels are increased in patients with psoriatic arthritis and their siblings. Rheumatology (Oxford). 2004;43:272–6. 16. Ren XH, Peng XD, Wu XP, Liao EY, Sun ZQ. Association between serum soluble membrane type matrix metalloproteinase1 (MT1-MMP) levels and bone mineral density, and biochemical markers in postmenopausal women. Clin Chim Acta. 2008;390: 44–8.

3369 17. Gallego R, Codony-Servat J, Garcia-Albeniz X, Carcereny E, Longaron R, Oliveras A, et al. Serum IGF-I, IGFBP-3, and matrix metalloproteinase-7 levels and acquired chemo-resistance in advanced colorectal cancer. Endocr Relat Cancer. 2009;16: 311–7. 18. Trivedi V, Boire A, Tchernychev B, Kaneider NC, Leger AJ, O’Callaghan K, et al. Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site. Cell. 2009;137:332–43. 19. Fosang AJ, Last K, Fujii Y, Seiki M, Okada Y. Membrane-type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain. FEBS Lett. 1998;430:186–90. 20. Kajita M, Itoh Y, Chiba T, Mori H, Okada A, Kinoh H, et al. Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J Cell Biol. 2001;153:893–904. 21. Mimori K, Fukagawa T, Kosaka Y, Ishikawa K, Iwatsuki M, Yokobori T, et al. A large-scale study of MT1-MMP as a marker for isolated tumor cells in peripheral blood and bone marrow in gastric cancer cases. Ann Surg Oncol. 2008;15:2934–42.